Dysferlin facilitates the de novo biogenesis of tubular membranes in hypertrophic remodelling

Abstract

Abstract Introduction Ferlins are transmembrane proteins with multiple C2 domains that may mediate Ca2+ dependent membrane fusion and repair events. In ventricles, Dysferlin expression is dominant, and patient mutations have been associated with cardiomyopathies. Hence, we hypothesized that Dysferlin is essential for the functional integrity of the tubular endomembrane system in ventricular myocytes (VM), and may facilitate the cellular hypertrophic remodelling in pressure-overload. Results In 30 weeks old Dysferlin knockout (KO) mice, echocardiography revealed dilative cardiomyopathy with reduced left-ventricular ejection fractions (difference between means KO vs. wildtype (WT) −13.45±2.17%, p<0.001, n=16/20 mice). Asking for the subcellular relevance of Dysferlin, we investigated VM nanodomains by immunolabelling in STimulated Emission Depletion (STED) nanoscopy and visualized punctual Dysferlin clusters decorating the T-tubule system of mouse and human VM. Precisely, Dysferlin clusters localized to junctions of the tubular system and the sarcoplasmic reticulum (SR) in nanometric proximity to RyR2 Ca2+ release units. Complexome profiling (LC-MS/MS) also suggested interactions of Dysferlin with RyR2 and the SR membrane-tethering protein Junctophilin-2 (JP2) in high molecular weight complexes. In JP2-overexpressing mice, live-cell STED membrane imaging captured polyadic junctional membrane structures in VM, specifically surrounded by dense Dysferlin clusters, which proposes Dysferlin as stabilizing protein of tubule-SR junctions. Further, we studied the ultrastructural remodelling of the tubular system in a mouse model of cardiac hypertrophy and heart failure induced by transaortic constriction (TAC). Interestingly, mortality four weeks post TAC was not increased in KO mice. However, echocardiography uncovered that Dysf-KO prevents left-ventricular hypertrophy in response to pressure-overload, which was confirmed by single cell measurements (VM area: WT 3559±120 μm2 vs. KO 2671±87 μm2, n=163/158 VM, p<0.001). Thus, we assumed Dysferlin to mediate tubular network proliferation during hypertrophic remodelling. Indeed, WT VM showed an augmented Dysferlin expression post TAC (158±0,125%, p=0,005, n=7/7 mice), and immunofluorescence STED nanoscopy detected an increased Dysferlin network complexity. In detail, we visualized highly abundant Dysferlin clusters decorating newly shaped axial tubules, which were quantified by component-specific tubule network analysis. Conclusion Nanoscale imaging proposes Dysferlin as stabilizer of tubule-SR junctions, thereby promoting functional excitation-contraction coupling, and demonstrates the crucial importance of Dysferlin in pressure-overload induced hypertrophic remodelling, facilitating the de novo biogenesis of axial tubule membranes and tubule-SR junctions necessary for VM growth. Thus, Dysferlin may emerge as potential novel therapeutic rationale to control subcellular membrane remodelling in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Multiscale Bioimaging Cluster of Excellence: From Molecular Machines to Networks of Excitable Cells;German Research Foundation (DFG) - Collaborative Research Centre 1002 (SFB 1002): Modulatory Units in Heart Failure